p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-containing enzyme that catalyzes the hydroxylation of aromatic compounds. In order to efficiently activate the phenolic substrate for hydroxylation by the flavin hydroperoxide, the enzyme lowers the phenolic pKa by about 2 units, a result (at least in part) of the interaction of the ligand phenolic oxygen with a tyrosine hydrogen bonding network. ITC will be used to investigate ligand deprotonation upon binding to several PHBH forms, including WT. Titrations will be conducted over a range of pH values, yielding the pH dependence of the thermodynamic parameters of binding. At any particular pH, titrations will be conducted using buffers which have different enthalpies for deprotonation. Thus, if deprotonations (or protonations) are important components in ligand binding, binding will be coupled to buffer protonation (or deprotonation), and different thermodynamic parameters will be obtained with different buffers at the same pH. An important control, differentiating between ligand deprotonation and the possibility of a currently unsuspected protein deprotonation linked to ligand binding, will be titrations with ligands lacking an ionizable group, such as p-aminobenzoate or benzoate. Several mutants exist that alter the pKa of bound pOHB (Tyr201Phe, Tyr385Phe, Asn300Asp, Arg220Lys, Lys297Met, His72Asn), and these will be studied in order to further our understanding of how PHBH effects the deprotonation of pOHB.